The design of efficient vaccines against infectious diseases remains a major challenge in medical science. Low cost, non-invasive administration, life-long protection by single doses combined with ease of preparation, storage and transport are desirable goals to be achieved. In this respect, live attenuated bacterial carriers that express heterologous antigens are attractive vehicles for the oral delivery of vaccines. This type of delivery should result in a broad spectrum of both mucosal and systemic immune responses. Use of vaccine vectors overcomes some of the limitation of oral delivery of proteins, which usually need to be co-administered with adjuvant proteins such as cholera toxin to evoke an immune response (Brown et al., 1987; Flynn et al, 1990). In addition, administration of live replicating vectors might be advantageous over other forms of administration such as microencapsulation because of the immunomodulatory properties of cell wall components of bacteria. Finally, the natural route of entry could prove to be of benefit since many bacteria like Salmonella egress from the gut lumen via M cells into Peyer's Patches (Jones et al, 1994; Neutra et al, 1996; Siebers and Finley, 1996) and migrate eventually into lymph nodes and spleen, thus allowing targeting of vaccines to inductive sites of the immune system.
Genetic immunization has recently provided a promising new approach to the vaccination problem (for review see Donnelly et al., 1997). Isolated plasmid DNA—introduced into muscle or skin of the host—leads to expression of antigen in the host cells when transcription is driven by eukaryotic control elements. This has led to B and T cell stimulation and to protective responses. How these responses are generated remains still unclear. Muscle cells apparently express low levels of MHC class I but lack MHC class II and costimulatory molecules. Although, it is not known which cells function as antigen presenting cells (APC) under these circumstances, it is likely that resident dendritic cells or macrophages capture the antigen and migrate to lymph nodes and spleen to stimulate CD4+ and CD8+ T cells. Indeed antigen expressing dendritic cells have been observed after genetic immunization into the skin using a gene gun (Condon et al., 1996). It is not known whether DNA is also transferred directly into dendritic cells when plasmids are applied into muscles.
Several advantages have been observed with genetic immunization over conventional vaccination. The DNA can be detected for a considerable period of time thus acting like a depot of antigen (Ning et al, 1993). Sequence motifs in some plasmids are immunostimulatory and can function as adjuvant (Krieg et al., 1995; Messina et al., 1991; Yamamoto et al., 1992). Co-expression of cytokines enhance the response and offer the possibility of modulating the induction of an immune response into a desired direction (Zhignan et al, 1995; Geissler et al, 1997; Kim et al, 1997). However, several obstacles need to be overcome before general applicability can be achieved.
If it would be possible to deliver plasmids for genetic immunization with an attenuated bacterial carrier, the advantages and versatilities of both systems would be combined. In addition, the natural route of administration would deliver DNA to cell types which have specifically evolved to induce immune responses. Salmonella spp. are particularly suited for this purpose because of the extensive knowledge on the genetics and physiology of many strains. A large body of documentation exists on their utility as heterologous antigen carriers that are capable of inducing protective immune responses (Fairwether et al., 1990; Molina et al., 1990; Newton et al., 1989; reviewed Chatfield et al, 1994; Roberts et al, 1994). Also, safe attenuated strains of Salmonella are available and are already in use as vaccines in animal husbandry and man (Hassan, 1996; Steinbach, 1996; Fox, 1997; Germanier and Fürer, 1975). Finally, recombinant plasmids constructed in laboratory strains of E. coli can be directly introduced into Salmonellae without further manipulations.